Attenuated mutants of Salmonella enterica Typhimurium mediate melanoma regression via an immune response

Abstract

The lack of effective treatment options for an increasing number of cancer cases highlights the need for new anticancer therapeutic strategies. Immunotherapy mediated by Salmonella enterica Typhimurium is a promising anticancer treatment. Candidate strains for anticancer therapy must be attenuated while retaining their antitumor activity. Here, we investigated the attenuation and antitumor efficacy of two S. enterica Typhimurium mutants, ΔtolRA and ΔihfABpmi, in a murine melanoma model. Results showed high attenuation of ΔtolRA in the Galleria mellonella model, and invasion and survival in tumor cells. However, it showed weak antitumor effects in vitro and in vivo. Contrastingly, lower attenuation of the attenuated ΔihfABpmi strain resulted in regression of tumor mass in all mice, approximately 6 days after the first treatment. The therapeutic response induced by ΔihfABpmi was accompanied with macrophage accumulation of antitumor phenotype (M1) and significant increase in the mRNAs of proinflammatory mediators (TNF-α, IL-6, and iNOS) and an apoptosis inducer (Bax). Our findings indicate that the attenuated ΔihfABpmi exerts its antitumor activity by inducing macrophage infiltration or reprogramming the immunosuppressed tumor microenvironment to an activated state, suggesting that attenuated S. enterica Typhimurium strains based on nucleoid-associated protein genes deletion could be immunotherapeutic against cancer.

Keywords: S. enterica Typhimurium; anticancer; macrophage; melanoma; mutants.

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FIGURE 1

Bacterial growth and virulence of mutants. (A) Growth of the mutants and wild-type strain, monitored for 12 h by measuring the OD (λ 600 nm); (B) number of colony forming units (CFU) per ml of the mutants and wild-type strain monitored every hour. Growth curves were performed three times. The graph shows the mean ± SD of three independent experiments. (C) Virulence of mutants and wild-type strains in the Galleria mellonella model. Larvae were inoculated with 10⁴ CFU in the last proleg, and survival was observed for 96 h. (D) Electrophoretic profile of lipopolysaccharides (LPS). Polyacrylamide gel stained with silver to confirm bacterial LPS integrity. 1. Dual color molecular weight standard (Bio-rad); 2. Salmonella enterica Typhimurium 14028; 3. S. enterica Typhimurium 14028 ΔihfABpmi; 4. S. enterica Typhimurium 14028 ΔtolRA. 5. Pseudomonas aeruginosa (Δwzz). The graphs show representative values from three independent experiments.

FIGURE 2

Invasion, survival, and cytotoxicity of mutant strains in bladder carcinoma (5,637) and melanoma (B16F10) cells. Cancer cells were infected in vitro with mutant strains for 2 and 6 h at an MOI of 10:1, after which the ability of invasion (A) and survival (B) of the mutant strains in cancer cells was determined by measuring intracellular CFU. 14028 WT was used as a positive control. Percentage of viable 5,637 (C) and B16F10 (D) cancer cells after 24 h of bacterial strain infections. The results were normalized to those of the uninfected cells. Results are represented as mean ± SD of three independent experiments performed in triplicate wells. Statistical significance was determined by one-way ANOVA followed by Multiplex Comparison Test *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.0001.

FIGURE 3

Safety assessment of treatment with attenuated mutants of Salmonella enterica Typhimurium. (A) Experimental design to test the toxicity and safety of mutants in mice inoculated by the subcutaneous route. (B) Body weight of mice inoculated with attenuated mutants throughout the experiment. Weight of spleens (C) and livers (D) of mice inoculated with attenuated mutants. In the graphics, the error bars represent the average ± SD of 5 mice per group. Data are from one experiment representative of two independent experiments. Each experiment performed with 5 mice per group. Statistical significance was determined by one-way ANOVA followed by Multiplex Comparison Test *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.0001.

FIGURE 4

Antitumor efficacy of attenuated mutants of Salmonella enterica Typhimurium in a B16F10 subcutaneous tumor model. C57BL/6JUnib mice were inoculated with B16F10 cells (3 × 10⁶), subcutaneously in the dorsal flank region. When the tumor reached 100 mm³ (10–12 days after tumor cell inoculation), 10⁶ CFU of the ΔtolRA mutant, 10⁵ CFU of the ΔihfABpmi mutant or PBS was injected intratumorally (n = 7) or intraperitoneal (n = 5) once a week for 2 weeks (n = 7). (A) Tumor growth after starting treatments intratumorally. (B) Weight of mice during treatments intratumorally. Data reported in graphs A and B are average values ± SD. (C) Tumor size at the endpoint of the treatments intratumorally. (D–F) Kinetics of individual tumor growth in groups treated intratumorally with PBS, ΔihfABpmi mutant, and ΔtolRA mutant, respectively. Day one is considered when the tumor reaches 100 mm³. Two independent experiments were performed, using seven mice per group. (G) Tumor growth after starting treatments intraperitoneal. (H) Weight of mice during treatments intraperitoneal. Data reported in graphs (G, H) are average values ± SD. (I) Tumor size at the endpoint of the treatments intraperitoneal. (J–L) Kinetics of individual tumor growth in groups treated intraperitoneal with PBS, ΔihfABpmi mutant, and ΔtolRA mutant, respectively. Day one is considered when the tumor reaches 100 mm³. One experiment was performed, using five mice per group. Statistical significance was determined by one-way ANOVA followed by Multiplex Comparison Test *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.0001.

FIGURE 5

Histological analysis of normal tissues. Tumor-bearing mice were treated with 10⁵ CFU of ΔihfABpmi mutant or PBS, twice a week for 2 weeks. After 1 week of the last inoculation, the spleen, liver, lung, and kidney were collected for histological analysis using H&E staining of organ sections. Slight inflammation was observed in the liver and spleen of mice treated with ΔihfABpmi mutant. The asterisks indicate the megakaryocytes in the spleens and inflammatory foci in the liver. Scale bar: 200 μm for ×100 and 50 μm for ×400.

FIGURE 6

Immunophenotyping of macrophages infiltrated in B16F10 tumors. (A) Representative gating strategy to identify macrophage (F4/80+ CD11b+), M1 macrophage (F4/80+ CD80⁺) and M2 macrophage (F4/80+ CD206+). (B) Macrophage population and (C) Macrophage phenotype (M1 or M2 type) after intratumoral treatment with 10⁵ CFU of the ΔihfABpmi mutant or PBS in a murine model of melanoma (three mice per group). Results are presented as the mean ± SD (n = 6). Statistical significance was calculated by Student’s t-test. *p < 0.05; **p < 0.01.

FIGURE 7

Effect of the ΔihfABpmi mutant on gene expression. The relative level of RNA expression of Bax (A), IL-6 (B), VEGF (C), iNOS (D), Ki-67 (E), and TNF-α (F) genes. Tumor tissue from B16F10 tumor-bearing mice was collected after 4 days of an intratumoral injection with 10⁵ CFU of the ΔihfABpmi mutant or PBS (n = 6). Results are presented as the mean ± SD. Statistical significance was calculated by Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.0001.